Angiogenesis in canine adrenocortical
tumors
Gene expression profiles of Angiopoietin-1 and -2, vascular
endothelial growth factor and their receptors.
Miriam Kool, 0051470, 1-10-2008
Supervisors: Sara Galac, Hans Kooistra, Jan Mol
Index
Abstract
Introduction
Adrenocortical tumors and Cushing’s syndrome
Pathogenesis of adrenocortical tumors
Genes of interest
Introduction to this study
Aims and hypothesis
Material and methods
Tumor and normal adrenal material
RNA isolation and cDNA synthesis
Q-PCR for determining expression levels
Regular PCR for detection of Ang-2443
Quantification of the full length Ang-2 and Ang-2443
Quantification of Ang-2 on protein level
Data analysis
Results
Q-PCR for determining expression levels
Regular PCR for detection of Ang-2443
Quantification of the full length Ang-2 and Ang-2443
Quantification of Ang-2 on protein level
Conclusions
Discussion
Acknowledgements
References
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Abstract
This paper describes a study concerning the expression of angiogenesis-related
genes in cortisol secreting adrenocortical tumors in dogs. The aim of this study was
to determine which angiogenesis-related genes are significantly up- or downregulated in these tumors, when compared to normal adrenal tissue. Six genes,
known to be involved in both normal and tumor angiogenesis, were chosen to
evaluate: Angiopoietin 1 and 2, their receptor Tie-2, vascular endothelial growth
factor (VEGF) and its receptors VEGFR 1 and -2. For quantification of gene
expression on messenger RNA level, a quantitative RT-PCR was performed on
material of 31 adrenocortical tumors and 9 normal, healthy adrenals.
In addition to determining the expression levels of these genes, the presence of Ang2443, a splice variant of Angiopoietin 2, was also investigated. In dogs this splice
variant had not yet been described; therefore the aim was to investigate whether or
not this variant was present in dogs, and if so, whether an association with cortisol
secreting adrenocortical tumors could be proven. To achieve this, regular PCR and
quantitative RT-PCR were performed.
To investigate whether the differences in the expression of Ang-2, detected on
messenger RNA level, were also present on protein level, a Western blot experiment
was performed.
Q-PCR results showed no significant differences in expression levels of Angiopoietin
1, Tie-2 and VEGFR 2, and only minor changes in the expression levels of VEGF and
VEGFR 1. However, for Angiopoietin 2, expression analysis showed a significant upregulation in the tumor group when compared to normal adrenals. This upregulation was confirmed on the protein level by means of Western blot. The
presence of Ang-2443 was demonstrated in both normal adrenals and adrenocortical
tumors. As for the full length Ang-2, expression analysis showed that this splice
variant was significantly up-regulated in the tumor group. Additionally, a difference
in expression levels between adenomas and carcinomas was shown for Ang-2443,
with higher levels of up-regulation in the malignant tumors. Western blot results
confirmed these findings on protein level.
The results of this study therefore strongly indicate a role of Angiopoietin 2 in the
pathogenesis of canine adrenocortical tumors, whereas for the other genes in this
study such a role is not implicated by the results. More research is needed to
determine the nature of the role of Ang-2 and its splice variant Ang-2443 in the
pathogenesis of canine adrenocortical tumors.
3
Introduction
Adrenocortical tumors are one of the causes of Cushing’s syndrome; one of the most
common endocrine disorders in dogs. In man many studies have been performed
regarding the pathogenesis of these tumors, and among these, some studies that
identify genes up- or down-regulated in these tumors in comparison to normal
adrenal tissue. By means of microarray and quantitative PCR (q-PCR), many
differentially expressed genes have been identified in man, among which some
genes that are known to play a crucial role in tumor angiogenesis.
Because angiogenesis is one of the main factors that facilitate tumor growth, and
over-expression of genes involved in angiogenesis has been shown in many tumors
in both human and canine origin, we have chosen to direct our attention in this
study to the expression profiles of angiogenesis-related genes in canine
adrenocortical tumors.
Adrenocortical tumors and Cushing’s syndrome
Tumors of the adrenal cortex are relatively common in dogs. These ATs (ATs) can be
divided into groups based on their functionality and their malignancy. ATs may
either be functional or non-functional. Most frequently, functional ATs rise from the
zona fasciculata and secrete excessive amounts of glucocorticoids. An
aldosteronoma, a tumor arising from the glomerular zone, is also possible, but
rarely occurs. In a non-functional AT, the tumor cells do not produce any hormones;
this kind of AT is called an incidentaloma. The remaining part of this introduction
will focus on the functional cortisol secreting AT and the disease it causes in dogs.
Apart from the distinction between functional and non-functional ATs, these tumors
may also be divided based on their malignancy. In dogs, benign adenomas and
malignant carcinomas of the adrenal cortex seem to occur in equal frequencies1,2.
Functional ATs give rise to a complex of clinical symptoms called Cushing’s
syndrome, which is one of the most common endocrine disorders in dogs. The
symptoms in animals are caused by an excess of glucocorticoid secretion by the
adrenal cortices.
Regulatory mechanisms of the hypothalamic-pituitary-adrenal axis
For a good understanding of Cushing’s syndrome and of how ATs contribute to this
syndrome, a basic understanding of the regulatory mechanisms of the
hypothalamic-pituitary-adrenal axis is essential.
Glucocorticoids are synthesized from cholesterol in the adrenal cortex, which
consists of three different zones. The middle and inner zones (zona fasciculata and
zona reticularis) form one functional unit, and are responsible for the glucocorticoid
production. The production of glucocorticoids by the adrenal cortex is stimulated by
adrenocorticotropic hormone (ACTH). ACTH is secreted in episodic bursts from the
anterior lobe of the pituitary; the frequency and magnitude of these bursts are
4
under central nervous system control. Release of ACTH is stimulated by
hypothalamic hormones arginine-vasopressin (AVP) and corticotrophin releasing
hormone (CRH). Several mechanisms act together to exert control over the amount
of glucocorticoids produced. Stress, for example from illness or surgery, induces an
increase in the amount of AVP and CRH secreted by the hypothalamus, thus
increasing ACTH secretion. Immunological factors can also trigger ACTH release, by
producing cytokines that stimulate CRH secretion.
The major inhibiting factor in the regulation of glucocorticoid production is the
negative feedback mechanism. High levels of glucocorticoids in the blood have an
inhibiting influence on the hypothalamus -inhibiting CRH and AVP release- as well
as on the pituitary, inhibiting ACTH release. Glucocorticoids also have an inhibitory
effect on immune reactions, thus lowering the amount of cytokines produced and
creating a second negative feedback loop.
Effects of glucocorticoids on tissue level
Glucocorticoids exert their effects on tissue level by diffusing into the cytoplasm of
the target cell, and binding to cytoplasmic glucocorticoid receptors. The
glucocorticoid-receptor complexes are then transferred into the nucleus, where they
influence mRNA- and protein synthesis.
Glucocorticoids increase the synthesis of key enzymes in gluconeogenesis, thus
increasing the amount of gluconeogenesis on tissue level, while glucose metabolism
and uptake are decreased. Protein synthesis also decreases, leading to larger
amounts of free amino acids, which may serve as substrate for gluconeogenesis. Via
these mechanisms glucocorticoids cause an elevation of blood glucose
concentrations. Glucocorticoids also have a direct stimulatory effect on lipolysis. An
indirect effect resulting from the elevated blood glucose levels is an increase in the
amounts of insulin released from the endocrine pancreas. The increased insulin
levels promote lipogenesis and fat deposition, thus overruling the direct lipolytic
effect of glucocorticoids. On the longer term the continued increased insulin levels
lead to insulin resistance, which can be the cause of a secondary development of
diabetes mellitus.
Apart from the metabolic effects, glucocorticoids also affect the immune system in
an anti-inflammatory way. Some of these effects are mediated by lipocortin, a
glucocorticoid induced protein that inhibits phospholipase A2. This inhibition
results in a decrease in the production of leukotrienes, thromboxanes and
prostaglandins, which are important inflammatory mediators. Glucocorticoids also
have an inhibiting effect on the production of interleukins. The decreased
production of interleukins, in turn results in an inhibition of the recruitment of
neutrophils, monocytes and macrophages to an area of inflammation.
Types of hypercortisolism
Hypercortisolism, or Cushing’s syndrome typically develops in middle-aged to
elderly dogs; small breeds tend to be more frequently affected. In this disease the
regulatory mechanisms described above are disturbed, resulting in an over5
secretion of glucocorticoids. Apart from functional ATs, two other major causes of
hypercortisolism can be distinguished: pituitary- or ACTH-dependent
hypercortisolism and an iatrogenic form resulting from long-term corticosteroid
administration.
In about 15% of dogs with spontaneous Cushing’s syndrome, the glucocorticoid
excess is caused by a functional AT, producing large amounts of glucocorticoids.
This form is also known as ACTH-independent hypercortisolism. As mentioned
before, both adenomas and carcinomas occur, in equal frequencies. Because of the
negative feedback mechanism, the pituitary release of ACTH is suppressed, resulting
in low ACTH concentrations in the peripheral blood. The tumor cells are not
sensitive to this feedback mechanism, and keep producing large amounts of
glucocorticoids in spite of the low ACTH levels. However, the glucocorticoidproducing cells in the contralateral normal adrenal are normally sensitive to the low
concentrations of ACTH, resulting in a decreased glucocorticoid production by these
cells, which on the longer term will lead to atrophy of the normal adrenal gland.
In the remaining 85% of dogs with non-iatrogenic Cushing’s syndrome, the hypersecretion of cortisol is caused by a functional tumor in the pituitary gland, which
produces large amounts of ACTH, and is much less sensitive to the inhibiting
influence of the feedback mechanism. The increased secretion of ACTH results in a
continuous stimulation of the adrenal cortices, thus leading to hyper-secretion of
glucocorticoids, and on the longer term, adrenal hypertrophy. This form of
hypercortisolism is called pituitary-dependent hypercortisolism (PDH).
The iatrogenic form of Cushing’s syndrome is caused by long-term use of exogenous
corticosteroids in high doses. Because of the feedback mechanism, ACTH secretion
will decrease, thus decreasing the level of glucocorticoid production in the adrenal
cortices. On the long term this will lead to atrophy of the glucocorticoid producing
zones of the adrenal cortices. Because this study concerns the pathogenesis of
cortisol secreting ATs, the following discussion of clinical signs, diagnostics and
therapeutic options will focus on the ACTH-independent form of hypercortisolism.
Clinical signs and biochemical changes
Clinical and biochemical changes in animals with Cushing’s syndrome are diverse
and can be attributed to the actions of glucocorticoids as described above. Common
clinical signs associated with hypercortisolism are polyuria and polydipsia. These
symptoms are a result of the lowering effect of glucocorticoids on the secretion of
AVP by the hypothalamus, combined with an interference of glucocorticoids with
the actions of AVP on cellular level in the kidney. The combined gluconeogenic,
lipogenic and protein degrading effects result in polyphagia, centripetal obesity with
a markedly enlarged abdomen, muscular atrophy and muscle weakness, lethargy
and exercise intolerance. The effects of the glucocorticoid excess on the skin and
hair coat commonly include alopecia and thinning of the skin. Not every patient will
exhibit all of these clinical signs; the range of symptoms exhibited is different for
each individual patient.
6
Complications that may arise from hypercortisolism can be severe and include
secondary diabetes mellitus, systemic hypertension, pulmonary thromboembolism,
and congestive heart failure.
The excess of glucocorticoids also causes changes in biochemical and hematological
parameters. Biochemically, an increase in alkaline phosphatase (AP) is a very
common finding in patients with Cushing’s syndrome. This results from
glucocorticoid-induced elevation of a heat stable iso-enzyme of alkaline
phosphatase: AP65. Other common biochemical changes include mildly decreased
thyroxin (T4) levels, and increased plasma levels of cholesterol, lipids and glucose.
Hematological changes in patients may include a decrease in the amounts of
lymphocytes and eosinophils, neutrophilic leukocytosis and mild erythrocytosis.
Establishing a diagnosis
Based on the previously mentioned clinical signs, biochemical and hematologic
changes, a presumptive diagnosis of hypercortisolism can be established. If there is
no history of corticosteroid administration, the spontaneous forms of Cushing’s
syndrome remain as the only options.
To confirm this presumptive diagnosis, and to determine whether the
hypercortisolism is of adrenal or pituitary origin, some specific tests are indicated.
Because of the episodic secretion of ACTH and glucocorticoids in normal individuals,
a simple measurement of cortisol in plasma is not sufficient to establish a diagnosis:
if measured during an episode of inactivity these values may be within reference
ranges in hyperadrenal dogs. Fortunately other tests are available, that can be used
apart or in combination to confirm a presumptive diagnosis of Cushing’s syndrome,
and to determine the origin. The diagnosis of hypercortisolism can be confirmed by
the urinary cortisol/creatinine ratio or the low dose dexamethasone suppression
test. For differentiation between the two forms of the disease, the high dose
dexamethasone suppression test can be performed and/or the basal plasma ACTH
concentration can be measured.
The laboratory test most commonly used in the Netherlands to diagnose
hypercortsolism is the urinary cortisol/ creatinine ratio. This test solves the
problem of episodic secretion, by measuring the cortisol excretion over a longer
period of time, and relating it to the urinary creatinine concentration. That makes
this test a reliable parameter of the glucocorticoid production in the adrenal
cortices. A second advantage is the possibility to combine this test for baseline
glucocorticoid secretion levels, with the high-dose dexamethasone suppression test,
to discriminate between pituitary and adrenocortical forms of the disease. The
principle behind this, is the fact that most pituitary tumors are less sensitive to the
inhibitory influence of glucocorticoids, but not completely insensitive. In other
words, the ACTH secretion of a pituitary tumor cannot be suppressed by normal
levels of glucocorticoids, but can be suppressed by administering a very high dosage
of corticosteroids. In contrast, an adrenal tumor will show no reaction at all to even
7
a high dosage of corticosteroids, being completely independent of ACTH stimulation,
and thus insensitive to the ACTH mediated feedback mechanism.
Thus, by administering a high dose of dexamethasone and measuring the effect on
the urine cortisol/creatinine ratio, a distinction can be made between PDH and AT.
In dogs with an AT no decrease in cortisol concentrations will occur, whereas in
most cases of PDH the cortisol/creatinine ratio will show a decline of 50% or more.
In some cases of PDH however, cortisol values do not decline in response to
dexamethasone administration, so in patients that show less than 50% decline other
tests are needed to discriminate between an AT and an inhibition resistant form of
PDH.
The low-dose dexamethasone suppression test is a simple screening test for
diagnosing hypercortisolism. This test is based on the resistance of ATs and the
relative resistance of PDH to suppression by the negative feedback mechanism,
resulting in a marked decline in cortisol concentrations in normal dogs, as opposed
to a lack of suppression in the hyperadrenal patient. The last relevant test is the
measurement of endogenous ACTH levels in plasma. In dogs with PDH, the ACTH
levels will be elevated, since the pituitary tumor causing the disease produces high
amounts of ACTH. In dogs with an AT however, the elevated plasma glucocorticoid
levels will result in a suppression of pituitary ACTH release through the negative
feedback mechanism.
In addition to these laboratory tests, diagnostic imaging can also contribute to the
establishment of a diagnosis. Useful techniques in case of an AT include abdominal
ultrasonography, thoracic radiography and computed tomography (CT). An AT
usually presents as an adrenal mass of variable size and shape on the ultrasound.
Because of the negative feedback mechanism, atrophy will have developed in the
contralateral adrenal gland, which will therefore be small or undetectable. When
performing ultrasonography, a careful examination of the liver for the presence of
metastasis and a check for invasion of the tumor into adjacent tissues or blood
vessels are other important aspects. Lately, CT has proven to be the most reliable
method to visualize adrenals and detect metastasis in the lungs.
Therapeutic options
Once the diagnosis has been established, therapy can be started. In case of an AT the
treatment of preference is adrenalectomy. The affected adrenal is completely
removed, thus removing the source of the excess glucocorticoid production. If the
tumor is located only in one adrenal gland, as is usually the case, adrenalectomy
results in a complete cure. If no metastases were present at the time of surgery, the
prognosis after adrenalectomy is good; however, dogs with metastases have a poor
prognosis. Because of the atrophy of the contralateral adrenal, glucocorticoid
supplementation is initially necessary. In the course of a few months,
supplementation can be gradually decreased and discontinued when the
contralateral adrenal has reached normal functionality. In case of a bilateral tumor,
8
bilateral adrenalectomy is an option. After this procedure lifelong supplementation
therapy of both glucocorticoids and mineralocorticoids is imperative.
If the adrenal tumor cannot be safely removed, for instance if invasion in
surrounding tissues and vessels has occurred, or if the condition of the patient does
not allow surgery, the only remaining treatment option is the use of Lysodren (o,p`DDD, Mitotane), a chemical agent which destroys the zones of the adrenal cortex.
Lysodren is also the treatment of choice when metastases are present. Although ATs
seem to be less sensitive to Mitotane treatment than the hypertrophic adrenals
observed in PDH, treatment with Mitotane may still achieve good results. The most
important complication of this therapy is the development of iatrogenic
hypoadrenocorticism. Therefore, supplementation with glucocorticoids, mineralocorticoids and salt is needed. In most cases, lifelong therapy with Lysodren,
including the supplementation therapy, is necessary.
9
Pathogenesis of adrenocortical tumors
In contrast to the extensive knowledge on the pathogenesis of Cushing’s syndrome
caused by ATs, little information exists on the pathogenesis of these tumors
themselves. Much is still unknown, for example on which molecular and genetic
changes precede neoplastic transformation of adrenocortical cells and which factors
influence tumor growth rate, cell differentiation, invasion into adjacent tissues or
vessels and metastasis.
One of the major factors known to be involved in the pathogenesis of many tumors
is angiogenesis. For a tumor to grow beyond a certain size, the formation of new
blood vessels within the tumor is needed, to provide the tumor cells with the oxygen
and energy necessary for growth. Angiogenesis is also involved in facilitating
metastasis: the newly formed blood vessels are often less well organized then
normal blood vessels, which makes it easier for the tumor cells to gain access to the
vascular network, en thus metastasize to distant tissues.
Because of the importance of angiogenesis in tumor growth and metastasis, much
research has been done to determine how tumor angiogenesis is regulated. Two
major groups of genes were identified, which are crucial in both normal and tumor
angiogenesis: the Angiopoietin family and the VEGF family. Both families consist of a
number of different ligands and receptors; of the Angiopoietin family, Angiopoietin
1 and 2 and receptor Tie-2 are the major players in both normal and tumor
angiogenesis. Of the VEGF family, this holds true for VEGF-A (usually just termed
VEGF) and VEGF receptor 1 and 2.
Much is already known about the role of these genes in regulation of angiogenesis in
the normal individual, for instance in embryonic vascular development and vascular
remodeling. Furthermore, all of these genes have been implicated in the
pathogenesis of several tumor types in both human and canine patients, for instance
showing increased expression levels, correlation to increased vessel density, poorer
prognosis or less differentiated cells. On their role in the pathogenesis of ATs,
however, only little is yet known. Studies using a combination of micro-array and
quantitative PCR to determine expression levels of different genes in human ATs,
have shown over-expression of Ang-23-5. Also, studies concerning expression of the
Angiopoietin and the VEGF family members in mouse adrenal cortex have shown
expression of all of these genes in the normal adrenal gland55,56.
These data provide some preliminary evidence for the involvement of these genes in
adrenocortical pathology. To my best knowledge no information has yet been
published on the involvement of the VEGF and Angiopoietin families in the
pathogenesis of ATs in dogs. Because of the crucial role of angiogenesis in tumor
development, and the known involvement of the Angiopoietin and VEGF families in
tumor angiogenesis, these two gene families are the focus of attention of this study.
In the following section each of the six genes of interest is introduced, going over its
functions and expression levels in the healthy individual and what is known about
the functions and expression levels in tumor development. Based on these data, a
hypothesis is formulated regarding the expected behavior of each of the genes in the
ATs evaluated in this study.
10
Genes of interest
Vascular Endothelial Growth Factor
Vascular endothelial growth factor (VEGF) is an angiogenic growth factor, which
binds to specific endothelial receptors; VEGFR 1 and 2. It is a crucial factor in the
early embryonic development of blood vessels, and plays a role in both
vasculogenesis and angiogenesis6,32,34. A null mutation on the VEGF gene results in
early embryonic death caused by defects in endothelial cell development,
angiogenesis and hematopoiesis, both in homozygous and in heterozygous mice 34,35.
In the postnatal period, VEGF continues to play an important role, for instance in
organ development and skeletal growth33,35.
In the adult individual, VEGF provides an anti-apoptotic signal to endothelial cells,
stimulates proliferation and migration of these cells and increases vascular
permeability9,12,32,33. VEGF expression has been demonstrated in virtually all
vascularized tissues in the healthy adult individual, with the highest levels found in
lung alveoli, renal glomeruli and adrenal cortex35,36,43. Interestingly, in mice the
expression of VEGF in the adrenal cortex was found to be down-regulated in
response to corticosteroid induced hypoadrenocorticism, an effect that in mice
could be reversed by supplementing ACTH56.
Regulation of VEGF expression levels involves up-regulation by several different
factors, including hypoxia, several growth factors, inflammatory cytokines and
thyroid hormone31,33,35. VEGF-induced angiogenesis plays a role in the vascular
remodeling, which is present in many physiological and pathological processes, for
instance in the female reproductive cycle, hair growth, wound healing, ischemiainduced collateral formation, endometriosis, rheumatoid arthritis, psoriasis and
tumor growth32.
VEGF has been proven to stimulate tumor angiogenesis in vivo and VEGF-negative
cells exhibited a strongly impaired ability for tumor growth in nude mice32,34. Apart
from its role in stimulating tumor angiogenesis, VEGF may also provide an autocrine
survival signal for tumor cells, thus contributing to tumor survival and growth32,50,51.
Increased expression and secretion of VEGF by both tumor cells and infiltrating
immune cells has been demonstrated in a variety of human tumor types, including
mammary carcinoma, hemangiosarcoma, lung cancer and lymphoma. In many of
these tumor types, increased VEGF expression is associated with increased
vascularity, metastasis, chemo-resistance and a poorer prognosis16,21,31,38. In dogs
several studies regarding VEGF expression in diverse tumor types have been
performed, showing increased VEGF expression in for instance mammary tumors,
seminomas, squamous cell carcinomas and lymphomas13,38-40,42. In addition,
increased expression of VEGF was linked to a poorer prognosis in dogs with various
spontaneous tumors, receiving radiation therapy. Higher levels of VEGF expression
tended to be present in the more aggressive and malignant tumor types, with less
differentiated cells and higher vascularity39-41.
11
In summary, VEGF is a strong promoting factor of angiogenesis, which plays
important roles in both embryonic and adult vasculature. It has also been shown to
be an important factor in tumor development, both by stimulating tumor
angiogenesis, and by directly stimulating tumor cell survival and growth. In many
tumors increased expression has been demonstrated, often in combination with
negative prognostic features.
Because of its up-regulation in numerous tumor types, and its correlation to
negative prognostic features in many of these tumors, in the present study, an upregulation of VEGF in the ATs was expected. Within the tumor group, the adenomas
were expected to show a lesser degree of up-regulation of this gene than the
carcinomas.
VEGF receptor 1
VEGF receptor 1 (VEGFR 1), or Fms like tyrosine kinase 1 (Flt-1), is the first of the
two endothelial receptors for VEGF. The exact role of this receptor is still subject to
discussion. It binds VEGF with a high affinity, but has low tyrosine kinase activity,
which gives rise to the theory of it being predominantly a decoy receptor,
preventing binding of VEGF to the more active VEGFR 2. In embryonic development
VEGFR 1 indeed seems to act as a negative regulator of VEGF activity, as shown by
the excessive proliferation of angioblasts and lack of vessel organization, resulting
in early embryonic death, in VEGFR 1 null mice44. However, under certain conditions
VEGFR 1 signaling does have a stimulatory role, promoting endothelial cell survival,
growth and migration, angiogenesis and vascular permeability32,33,44. Possibly,
receptor interactions with VEGFR 2 are involved in determining whether VEGFR 1
has a positive or negative regulatory effect under given conditions45. Apart from its
roles as both negative and positive regulator in endothelial cells, VEGFR 1 signaling
is also involved in hematopoiesis, monocyte and macrophage migration and
development of osteoclasts, osteoblasts and the bone marrow cavity33,35,44,46.
VEGFR 1 expression in the healthy adult individual was detected in a number of
normal cell- and tissue types including endothelial cells, monocytes, macrophages,
smooth muscle cells, gastrointestinal epithelium and osteoblasts32,44,47,48. Expression
was also shown in the adrenal cortex of the mouse, but was not sensitive to
inhibition by iatrogenic hypoadrenocorticism56. VEGFR 1 was shown to respond to
hypoxia and macrophage activation with a rise in expression levels33,44.
Apart from their roles in the normal physiology, the VEGF receptors have been
implicated in many pathological situations, for instance in tumor development,
stimulating tumor growth, angiogenesis, metastasis and ascites formation, but also
in inflammatory diseases such as arthritis or psoriasis. VEGFR 1 expression was
found to be increased in several human tumor types, including colorectal tumors,
leukemia’s, renal and mammary carcinomas32,33,37,49. In the study on expression of
VEGF and its receptors in canine lymphomas, VEGFR 1 expression was detected in
54% of lymphomas, in variable amounts, with high levels of expression in 23% of
the investigated tumors38.
12
In summary, VEGFR 1 is a VEGF receptor with both negative and positive regulatory
functions, which plays a role in various pathological conditions, including tumor
development. Its expression is increased in a number of different tumor types.
Because VEGFR 1 has both a negative and a positive regulatory role in angiogenesis,
the formulation of a hypothesis regarding its behavior in canine ATs is difficult.
However, because of the known up-regulation of this gene in several human tumor
types, and the implicated role of this receptor in the pathogenesis of many
pathological situations, an up-regulation of this gene in the ATs, seems the most
likely outcome of this study.
VEGF receptor 2
VEGF receptor 2 (VEGFR 2), or kinase insert domain receptor (KDR), is the other
endothelial receptor for VEGF. Despite its lower affinity for VEGF, this receptor is
the primary mediator of VEGF signaling32,33. Binding of VEGF to this receptor
promotes endothelial cell survival, proliferation and migration, vessel formation and
an increase in vessel permeability33. Mice lacking this receptor show a complete lack
of endothelial cell development, a complete absence of vasculogenesis and strongly
impaired hematopoiesis, resulting in early embryonic death 8,32,33.
In the healthy adult individual, expression of VEGFR 2 has been detected in
endothelial cells of almost all vascularized tissue types, osteoblasts, neuronal cells
and vascular smooth muscle32,43,44,47. In mice, expression of VEGFR 2 was
demonstrated in the adrenal cortex, and VEGFR 2 levels showed a decrease in
response to corticosteroid induced hypoadrenocorticism56. Expression levels of
VEGFR 2 have been shown to increase in response to hypoxia and high levels of
VEGF50,51.
Apart from their roles in the normal physiology, the VEGF receptors have been
implicated in many pathological situations, for instance in tumor growth. Signaling
by VEGFR 2 may contribute to tumor pathology both in a paracrine way, stimulating
angiogenesis, and in an autocrine way, stimulating tumor cell survival and
growth50,51. In man, increased VEGFR 2 expression has been shown in several tumor
types including lymphomas, lung-, renal- and mammary carcinomas, breast- and
gastric cancer32,37,52,53. In the previously mentioned study on the expression of VEGF
and its receptors in canine lymphoma, only 28% of tumors showed VEGFR 2
expression, however, the levels of expression were markedly higher than those in
normal lymph nodes38. Canine mammary tumors were also shown to express VEGFR
2, the levels of expression increasing significantly with malignancy and a less
differentiated cell type50,51.
Resuming, VEGFR 2 is the primary mediator of VEGF signaling and binding of VEGF
stimulates angiogenesis and vascular permeability. It has been implicated as a factor
in the pathogenesis of a variety of tumor types, and increased expression in many
tumor types has been demonstrated.
Because of its function as the primary mediator of the angiogenesis promoting effect
of VEGF, and because of its role in the pathogenesis of various tumor types, an upregulation of VEGFR 2 in the canine AT’s investigated in this study was expected.
13
With regard to the tumor grade, a higher degree of up-regulation in the carcinomagroup was expected.
Angiopoietin 1
Angiopoietin 1 is the primary agonist for the Tie-2 receptor. It plays a crucial role in
embryonic vascular development, in which it induces vessel sprouting and
remodeling, mediates endothelial cell interactions with, and adherence to,
surrounding support cells and is involved in proper development of the cardiac
trabeculations6,8. A knockout study showed that Ang-1 negative mice die early in
embryonic development, due to defects in angiogenesis, heart development and
vessel integrity, whereas over-expression of Ang-1 leads to development of larger
amounts of vessels, displaying more branching and a larger diameter8,55.
In the adult individual Ang-1 decreases vascular permeability, protecting against
vascular leakage, acts as an anti-inflammatory agent, provides a chemotactic signal
for endothelial cells and promotes stabilization of vascular networks by inhibiting
endothelial cell apoptosis6,7,9,10-12. Expression of Ang-1 has been demonstrated in
normal adult vasculature, as well as in a wide range of normal adult tissues
including skeletal muscle, ovary, uterus, placenta and prostate. Expression was
shown to decrease in response to certain inflammatory mediators, for instance
TNFα and IL-1β11,31. In healthy adult mice expression of Ang-1 in the adrenal cortex
had been demonstrated. Interestingly, expression levels of Ang-1 were found to be
down-regulated in corticosteroid induced hypoadrenocorticism in mice55. In canines
a study has been performed determining expression levels of Ang-1 and 2 and VEGF
in spontaneous tumors and normal tissues. In this study Ang-1 was found to be
expressed in all normal tissues examined, including the adrenal cortex; highest
expression levels were found in lung tissue, skeletal muscle and small intestine13.
On the expression and role of Ang-1 in tumors, contradicting information exists.
Over-expression of Ang-1 has been demonstrated in acute and chronic myeloid
leukemia’s, glioblastomas and several breast cancer cell lines. In contrast, artificial
over-expression of Ang-1 in a different a human breast cancer cell line, led to a
reduction in tumor growth14,29,30. In a number of other studies no significant
differences in expression levels were found between normal tissue and tumors. In
the previously mentioned study on Ang-1 expression in canine tissues and tumors,
over-expression was present in only one of the seven investigated tumors, a
mammary spindle cell carcinoma11. To my knowledge no other studies involving
Ang-1 expression levels in canine ATs have yet been performed.
In summary, Ang-1 is a stabilizing factor on the vascular endothelium, which acts
through its receptor Tie-2 to regulate embryonic angiogenesis, and is expressed in
most adult tissue types. Its role in tumor development is still unclear, as studies on
this subject showed contradicting results.
Because of the contradicting results of studies addressing the expression of Ang-1 in
tumors, a hypothesis regarding the expected behavior of this gene in canine ATs was
difficult to formulate. However, in most tumors with active angiogenesis a
destabilized vascular endothelium is observed, often in combination with increased
14
vascular permeability, whereas Ang-1 promotes the opposite effects. Therefore we
hypothesize that in the present study, Ang-1 will be down-regulated in canine ATs,
when compared to normal adrenocortical tissue. We expect this down-regulation to
be present to a greater extend in the malignant tumors.
Angiopoietin 2
Angiopoietin 2 is a competitive antagonist of Ang-1, which binds to the same
common receptor, Tie-2, but does not induce receptor phosphorylation. Ang-2
antagonizes the stabilizing effect of Ang-1 on the vascular endothelial cells, leading
to a more plastic, unstable state of these vessels. In the presence of VEGF,
subsequent angiogenesis is facilitated, as shown by an increase in capillary
diameter, proliferation and migration of endothelial cells and sprouting of new
vessels. However, in the absence of VEGF, apoptosis and subsequent vessel
regression will occur15. During embryogenesis Ang-2 is involved in vascular
development as a functional antagonist of Ang-1. Artificial Ang-2 over-expression
during this developmental period leads to similar abnormalities as seen in Ang-1
null-mice. Ang-2 may also play a role in embryonic lymphatic system
development23.
In the healthy individual, Ang-2 is present in high concentrations at sites of vascular
remodeling, for instance in areas of wound healing and in the female reproductive
tract6,15,55. Ang-2 expression increases in response to hypoxia; high levels of VEGF
also result in an increase of its expression31. In a study investigating Ang-1 and Ang2 expression levels in normal canine tissues and 7 spontaneous canine tumors, it
was shown that like Ang-1, Ang-2 is expressed in all normal canine tissues, including
the adrenal glands. Highest levels of expression were found in skeletal muscle, lung
tissue and small intestine13.
Because of its destabilizing effect on the vascular endothelium, which in presence of
VEGF leads to angiogenesis, it seems likely that Ang-2 might also play a role in
tumor angiogenesis. Over-expression of Ang-2 has indeed been demonstrated in
many human tumors, including hepatocellular carcinomas, gastric carcinomas,
squamous cell carcinomas, glioblastomas and lung carcinomas16-20,22,30.
Furthermore, increased expression of Ang-2 was significantly correlated with
increased vascular involvement, more advanced tumor stage and poorer prognosis
in several types of human tumors, including hepatocellular carcinomas, gastric
carcinomas, acute myeloid leukemia and lung cancer16,17,21,22. In these tumors, overexpression of Ang-2 was often seen in combination with VEGF overexpression17,19,22. Artificial over-expression of Ang-2 led to faster and more
aggressive tumor growth, with increased vascular involvement and hemorrhaging
in both gastric and hepatocellular carcinomas implanted in mice17,18.
Microarray of human ATs has demonstrated an increased expression of Ang-2 in
these tumors, which was confirmed and quantified by means of q-PCR3-5. In the
previously mentioned study on Ang-1 and 2 expression levels in canine tissues and
tumors, increased levels of expression were detected in both of the investigated
mammary simple carcinomas, in the splenic hemangiosarcoma and the
15
hepatocellular carcinoma13. To my knowledge no other studies involving Ang-2
expression levels in canine tumors have yet been performed.
Recently, a splice variant of Angiopoietin 2 has been indentified in man, which is
different from the original Ang-2 in missing exon 2. The alternatively spliced
isoform is secreted as a 443 amino acid long, glycosylated homodimeric protein and
is named Angiopoietin-2443 or Ang-2C. Like Ang-2, it binds to the Tie-2 receptor, but
does not induce receptor phosphorylation. Receptor binding of Ang-2443 inhibits
binding of Ang-1 and Ang-2 to the Tie-2 receptor, and also inhibits Ang-1 induced
receptor phosphorylation61,62.
In the study describing the isolation of Ang-2443, mRNA expression of this splice
variant was detected alongside of full length Ang-2 expression in endothelial cells,
as well as in several non-endothelial tumor cell lines and primary tumors such as
hemangioma, gastric carcinoma and breast carcinoma. In the latter case Ang-2443
mRNA expression was tumor specific; in the adjacent normal mammary tissue no
Ang-2443 mRNA could be detected61,63.
These results suggest that Ang-2443 might function as a competitive antagonist of
Ang-1 in the same way as Ang-2, and might play an important role in regulation of
angiogenesis, for example in neoplasia and inflammatory processes.
In summary, Ang-2 is the primary antagonist of Ang-1, which has a destabilizing
effect on the vascular endothelium, leading to either angiogenesis or vessel
regression, depending on the presence or absence of VEGF. It plays an important
role in tumor development as shown by its over-expression and correlation to
negative features in many tumor types.
Based on the existing knowledge on the functions and expression of Ang-2, our
hypothesis was that expression levels of Ang-2 would be raised in canine ATs. With
regard to the splice variant, our hypothesis is that Ang-2443 will be present in both
ATs and normal adrenals, but will show higher levels of expression in the tumors.
For both the full length Ang-2 and Ang-2443, a higher expression level in malignant
ATs was expected.
Tie-2 receptor
Tyrosine kinase receptor Tie-2 (or TEK) is the common receptor for the
Angiopoietin family, and binds both Ang-1 and Ang-2. Tie-2 regulated pathways play
a crucial role in embryonic development of blood vessels, regulating vessel
sprouting, remodeling and stabilization and promoting endothelial attachment to
the underlying supportive tissues7. Mice lacking Tie-2 die in an early embryonic
stage, showing incomplete cardiac development, widespread hemorrhage,
decreased numbers of endothelial cells, fewer and simpler vessels, and decreased
amounts of, and adhesion to, vessel support tissue7,23-25. Apart from its role in
embryonic angiogenesis, Tie-2 signaling also seems to be involved in embryonic
hematopoiesis, as hinted by the presence of this receptor on several hemopoietic
cell types23. Tie-2 deficient embryo’s have a pale and anemic appearance and lack
the ability to develop definitive hemopoietic cells24,26.
16
In the healthy adult individual, Tie-2 is present in endothelial cells and several
hemopoietic cells, and is up-regulated at sites of angiogenesis7,23,28,29. Expression
levels of Tie-2 rise in response to hypoxia and certain inflammatory cytokines, such
as TNFα and interleukin-1β31. In the adult individual, Tie-2 is involved in regulation
of both vascular stabilization and vascular remodeling. Persistent expression and Ang-1 induced- phosphorylation of Tie-2 provides a survival signal for endothelial
cells, preventing apoptosis, increasing vascular stability and decreasing vessel
permeability7,23. On the other hand, Ang-2 binding to the Tie-2 receptor promotes
detachment of the endothelial cells from surrounding support tissue. In the
presence of VEGF this detachment facilitates vessel sprouting, leading to new vessel
formation and vascular remodeling. However, in absence of VEGF this detachment
precedes apoptosis and vessel regression.
In mice, expression of Tie-2 in the adrenal cortex has been demonstrated, which,
like Ang-1, was down-regulated in response to corticosteroid induced
hypoadrenocorticism. Apart from its functions in regulation of vascular integrity,
Tie-2 signaling may also play a role in adult hematopoiesis28.
The up-regulation of Tie-2 in sites of angiogenesis, and the increase in its expression
levels in response to hypoxia and inflammatory mediators, suggest that Tie-2 might
also show increased expression in tumors, in which angiogenesis, hypoxia and
inflammatory mediators are frequently present. Indeed, Tie-2 mediated pathways
appear to play a role in tumor pathogenesis. In man, an increased expression of the
Tie-2 receptor has been demonstrated in a variety of tumors, including acute and
chronic myeloid leukemia’s, glioblastomas, mammary carcinomas, and gastric and
hepatocellular carcinomas14,17,27-30. High expression levels of Tie-2 were often
correlated with negative features, like less differentiated cells, increased tumor size
and cellularity and a poorer prognosis21,27. To my knowledge no studies have yet
been performed on the expression of Tie-2 in canine tumors.
Resuming, Tie-2 is the common receptor for Ang-1 and 2, and plays important roles
in both vascular stabilization and remodeling. Because of its actions, a role in tumor
angiogenesis seems plausible, and over-expression of this gene and a correlation to
poor prognosis and negative features have indeed been shown in many human
tumor types.
Based on the functions and expression data of Tie-2, a raised expression level of Tie2 in the canine ATs included in this study was expected. Furthermore, a higher
expression in the carcinomas was expected, when compared to adenomas.
17
Introduction to this study
From the previous descriptions of Ang-1 and 2, their receptor Tie-2 and VEGF and
its receptors, can be concluded that all of these genes have important functions in
both physiological processes involving angiogenesis and pathological situations. In
particular, a role in tumor pathogenesis has been implicated for all of them. A better
understanding of the functions, mechanisms of action and expression profiles of
these genes, in both healthy and diseased tissue, thus may contribute to a better
understanding of tumor pathogenesis. Furthermore, research into the role of these
genes in tumor pathogenesis may lead to the development of new diagnostic and
prognostic markers and ultimately, new therapeutic strategies.
Already, research into these genes in human medicine has led to the discovery of the
use of some of them as prognostic markers. Also, experimental therapeutic
strategies, for example blocking the VEGF signaling pathway, have shown promising
results. In veterinary medicine however, much less is known about the function of
these genes in tumor pathogenesis or their use in the development of prognostic
and diagnostic markers, whereas I could find no information at all on their use in
new therapeutic strategies.
So, in spite of the large amount of knowledge already present on these genes and
their functions, much also still remains to be discovered, especially in the veterinary
field. With this study we hope to gain more knowledge on the role of the
Angiopoietin and VEGF families in the pathogenesis of canine ATs.
18
Aims and hypotheses
The aim of this study was to determine which genes involved in angiogenesis, are
significantly up- or down regulated in ATs, as compared to normal adrenal tissue. A
comparison between adenomas and carcinomas is also made, to determine whether
there is a significant difference in expression levels between these two groups of
tumors.
In addition to determining the expression levels of these genes, we were interested
to know whether the splice variant of Angiopoietin 2, which has recently been
identified in humans, is also present in dogs. If this splice variant could be detected
in dogs, we were also interested in its expression levels, and in differences in
expression profiles between the ATs and normal adrenal tissue.
To determine the expression levels of the genes of interest on messenger RNA level,
a quantitative RT-PCR was performed on samples from 31 ATs and 9 normal
adrenal cortices. Six genes that play a prominent role in both normal and tumor
angiogenesis were investigated: Angiopoietin 1 and 2, their receptor Tie-2, VEGF
and its receptors VEGF receptor 1 and 2. For detecting the presence of the splice
variant of Angiopoietin 2, a regular PCR was performed on all of the samples, which
was designed to discriminate between the full length Ang-2 and the splice variant.
For quantification of the expression levels of both the full length Ang-2 and its splice
variant, a quantitative RT-PCR was performed, using primers specific for these two
isoforms. Additionally, a Western blot experiment was performed to determine the
amounts of Angiopoietin 2 protein present in the samples.
Based on present knowledge about the functions of the target genes, and what is
known about their role in tumor pathogenesis, a hypothesis was formulated
regarding the expected behavior of these genes in canine ATs. For Ang-2, Tie-2,
VEGF, VEGFR 1 and 2 an up-regulation of gene expression was expected in the
tumor group. Additionally, for Ang-2 we expect to confirm the presence of Ang-2443,
and to find higher expression of this splice variant in the tumors. For Ang-1 a downregulation in the tumor-group was expected.
19
Material and methods
Tumor and normal adrenal material
In this study 31 ATs (table 1) were examined, along with 9 samples of normal
adrenal tissue. Both adenomas and carcinomas were included. All ATs used in this
study were derived from patients presented to the university clinic for companion
animals at the faculty of veterinary medicine in Utrecht between 2001 and 2008.
After establishing a diagnosis, ATs were removed surgically and the tumor material
was stored at -70°C. After surgical removal, all tumors were subjected to histological
evaluation, to confirm the diagnosis and to determine whether an individual tumor
was benign or malignant. All of the tumors were recently evaluated by one
pathologist. A list of the tumors used in this study is depicted in table 1.
20
Number
Owner name
Tumor
classification
Patient nr.
Pathology nr.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Baljet
Gercke, van
Brouwer
Does
Doring
Reek, van de
Frederiks
Fischer
Geest, van
Groenedijk
Hazekamp
Keizer
Mooyman
Rooy
Struyk
Veen, van der
Verberk
Vermeulen
Verweel
Waldeck
Wilmes
Jellema
Jong, de
Ribbens
Ottens
Laar, van
Harms
Neumayer
Askeland
Vente
Dijkstra
A
C
A
C
C
C
C
C
A
A
C?
C?
A
A
C
C
C
C
C
C
A
A
A
A
C
A
A
C
C
A
A
501795
307649
221016
311316
228380
412393
219680
600914
306624
216001
601184
602408
304360
225696
216027
202841
H01.0223B
410807
228018
600227
400056
508028
603713
609558
700948
H01.3118.Y
H01.5333.C
506709
608328
702869
704792
P0503304
P0309551
P0207771
P0312916
P0300992
P0501181
P0207261
P953
P0308053
P0201932
P2319
P2534
P0307810
P0213365
P0201693
P0304310
P0101323
P0412680
P0214477
P1734
P0401610
P0701991
P070031
P0702209
P0200347
P0506625
P0609625
Table 1: This table lists the ATs used in this study, including their histological qualification, patientand pathology-numbers. In the “tumor classification” column, A stands for adenoma and C for
carcinoma. For four of the tumors, pathology numbers were not available, and only patient numbers
are listed.
Normal, healthy adrenals were derived from dogs belonging to the General animal
laboratory of Utrecht University. These dogs were euthanized for reasons other than
Cushing’s syndrome and their adrenal glands were removed after euthanasia. These
normal adrenal glands were also stored at -70°C, and were used in this study as a
control group.
21
RNA isolation and cDNA synthesis
RNA was isolated from the tissue samples using the RNeasy® mini kit, according to
the manufacturer’s protocols. An optional extra DNAse step was performed to
eliminate genomic DNA as much as possible. After RNA isolation, the total
concentration of RNA was measured using Nanodrop® technology. Samples
containing less than 100 ng/μl were excluded from the study. Subsequently cDNA
synthesis was performed using the iSCRIPT® cDNA synthesis kit, according to
manufacturer’s protocols. All RNA and cDNA samples were stored ad -20 °C.
Q-PCR for determining expression levels
To quantify the expression levels of the genes of interest on mRNA level, a
quantitative polymerase chain reaction (q-PCR) was performed on cDNA of all
samples. Primers for q-PCR were developed using the DNA-star® Primer Select
program and the M-fold® program. The following criteria were used to select
appropriate primer pairs:
- a product length between 100 and 200 base pairs
- no alternate bindings sites for the primers
- primers should span at least one intron, of preferably 3000 base pairs or
more
- preferably, primers should end on either C or G
- small difference in Tm of upper and lower primer
- product loop free temperature below optimal annealing temperature
For validation of the primers, the temperature range and optimal annealing
temperature were determined and PCR products were sequenced to determine
whether the correct product had been formed.
To determine the temperature range and the optimal annealing temperature, q-PCR
gradients were run for each of the primer pairs, with annealing temperatures
ranging from 55 to 65°C. One sample of normal adrenal tissue (M) was used for
running all of the gradients.
After completing the PCR gradient reactions, results were analyzed to find the
proper temperature range and the optimal annealing temperature for each of the
primer pairs. Criteria for the temperature range within which the primer pairs work
appropriately, were a reaction efficiency between 95 and 105% and a slope around 3,321. Within this range, the optimal annealing temperature was defined as the
temperature, at which the efficiency was closest to 100%,
To ascertain that the correct products had been formed, PCR products were
amplified in the Big Dye Cycle, and subsequently purified and sequenced. The
protocols for the Big Dye Cycle, purification and sequencing are added to this report
as appendices. For 5 out of 6 primer pairs, specific and correct products had been
formed. However, the primer pair for Ang-2 produced a second - aspecific - product,
therefore new q-PCR primers were developed using OligoExplorer®, a different
22
program for primer design. The same primer criteria were used as described for
primer design using the Primer Select program. However, no intron-spanning
primer pairs were detected by the program, so a non intron-spanning primer pair
was chosen. The procedure for validation of this new primer pair was identical to
the process for validation of the other primer pairs, as described above. After
sequencing, this primer pair was shown to deliver only the correct product,
therefore it was suitable for use in q-PCR.
For all of the primer pairs the sequences, appropriate temperature range, optimal
annealing temperature, positioning on the gene and product length are given (table
2).
Q-PCR primers
(Position)
Primer sequence
Optimal annealing
temperature
(Range)
Product
length
Angiopoietin 1
Upper primer
Lower primer
(926 -1087)
AAT AAT ATG CCA GAA CCC AAA AAG
CCC CAG CCA ATA TTC ACC AGA G
62 °C (61-63 °C)
162 bp
Angiopoietin 2
Upper primer
Lower primer
(1002-1171)
CGG CGT GAA GAT GGC AGT GTT G
GCC TCG TTT CCC TCC CAG TCC
57 °C (57-63 °C)
170 bp
Ang-2 2nd pair
Upper primer
Lower primer
(96-182)
AGA AGC ATG GAC AGC ATC GG
GTT GTC TGT TTC TGG CAG GAG G
64 °C (61,4-64,5 °C)
87 bp
Tie 2
Upper primer
Lower primer
(25-138)
CAG CTT ACC AGG TGG ACA TTT TTG
GTC CGC TGG TGC TTG AGA TTT AG
58 °C (57-64 °C)
104 bp
VEGF
Upper primer
Lower primer
(6-107)
CTT TCT GCT CTC CTG GGT GC
GGT TTG TGC TCT CCT CCT GC
58 °C
102 bp
VEGF R 1 (Flt 1)
Upper primer
Lower primer
(189-378)
GGC TCA GGC AAA CCA CAC
CCG GCA GGG GAT GAC GAT
63 °C (56-64 °C)
190 bp
VEGF R 2 (KDR)
Upper primer
Lower primer
(3606-3785)
GGA AGA GGA AGT GTG TGA CCC C
GAC CAT ACC ACT GTC CGT CTG G
64 °C
181 bp
Table 2: This table lists the primer pairs used for the target genes in this study, giving the sequences,
appropriate temperature ranges, optimal annealing temperatures, positioning on the gene and
product lengths.
23
To allow for normalization of gene expression, 4 reference genes were chosen, that
are known to be stably expressed throughout different tissue types, both in normal
and in tumor tissue. These genes were RPL8 (ribosomal protein L8), RPS5
(ribosomal protein S5), RPS19 (ribosomal protein S19) and HPRT (hypoxanthine
phosphoribosyltransferase). The expression levels of these reference genes were
also measured by q-PCR in all of the samples (Table 3).
Reference gene
primers q-PCR
Sequence
RPL8
upper
lower
CCA TGA ATC CTG TGG AGC
GTA GAG GGT TTG CCG ATG
RPS5
upper
lower
TCA CTG GTG AGA ACC CCC T
CCT GAT TCA CAC GGC GTA G
RPS19
upper
lower
CCT TCC TCA AAA AGT CTG GG
GTT CTC ATC GTA GGG AGC AAG
HPRT
upper
lower
AGC TTG CTG GTG AAA AGG AC
TTA TAG TCA AGG GCA TAT CC
Optimal annealing
temperature
55 °C
62,5 °C
61 °C
56 °C
Table 3: This table lists the primer pairs used for the reference genes, giving the sequences and
optimal annealing temperatures.
After validation of the primer pairs, q-PCR was performed for all of the genes of
interest and the reference genes, on both tumor- and normal samples. All reactions
were run at the optimal annealing temperature and according to the same protocol.
Regular PCR for detection of Ang-2443
To detect the presence of Ang-2443, PCR primers were developed to distinguish
between the two isoforms. These primers were developed using the DNA-star®
Primer-select program; primer conditions were the same as for development of the
q-PCR primers, except for the product length. The primers were designed to anneal
on either sides of exon 2, so that the full length Ang-2 will produce a long replication
product of 553 basepairs, whereas Ang-2443 should result in a PCR product that is
155 basepairs shorter, as a result of the deletion of exon 2. Primer locations and
expected PCR products are shown in a schematic diagram (figure 1). Primer
characteristics are listed in table 4.
24
Fig. 1: In this schematic diagram the primer locations (black arrows) are shown in both the full
length Angiopoietin 2 and the splice variant. The replication product is indicated by the thick red line.
Regular PCR
primers
(Position)
Primer sequence
Optimal
annealing
temperature
Ang-2
(106-658)
54 °C
Upper
ACA GCA TCG GGA GAA GGC AGT ATC
Lower
TCT TCT TTT ATT GAC CGT AGT TGA
Product length
553 bp
(full length)
398 bp
(splice variant)
Table 4: This table shows the characteristics of the primer pair used for detection of Ang-2443, giving
the primer sequences, appropriate temperature range, optimal annealing temperature, positioning
on the gene and product lengths.
To validate the primer set, a PCR was run on one of the normal samples (M), at the
optimal annealing temperature as given by the primer select program (54 °C).
Running the PCR at lower temperatures resulted in aspecific binding of the primers.
Because no PCR products were formed when using ordinary Taq polymerase,
platinum Taq was used. The resulting PCR products were separated by gel
electrophoresis, using a ladder to determine the length of the fragments. Two bands
were detected after gel electrophoresis of the test sample, with an approximate
length of 550 and 400 basepairs. Bands were carefully cut from the gel, and the DNA
was isolated from the gel using the QIAquick® gel extraction kit, and following the
protocol provided by the manufacturer.
After gel extraction, DNA concentrations of the extracted products were measured
using Nanodrop® technology. To determine the identity of the PCR products,
sequencing was performed, and the resulting sequence was blasted against the
canine genome. Sequencing results showed that the primers were working properly,
and the correct products had been formed.
The next step was to determine whether Ang-2443 was present in all of the samples
of both ATs and normal adrenals. To achieve this, a regular PCR was performed on
all of the samples, using the primers described above. PCR-products were
25
subsequently separated by gel electrophoresis, and the bands on the gel were
evaluated.
Quantification of the full length Angiopoietin 2 and Ang-2443
Evaluation of the bands after regular PCR showed the presence of both full length
Ang-2 and Ang-2443 mRNA in all of the samples, with varying band intensity. These
results were of course very promising; however, because no quantitative
information can be derived from this evaluation, a way to quantify the relative
expression levels of the full length Ang-2 and Ang-2443 was needed. To achieve this,
specific q-PCR primers were developed that would replicate only the full length
Ang-2 and the splice variant, respectively. These primers were developed using the
DNA-star® Primer Select program and the M-fold® program, using the same criteria
as mentioned previously. Because the program could not detect any primers specific
for the splice variant, that met all of these criteria, criteria for product length were
loosened to a maximum of 250 basepairs, after which a suitable primer pair could
be detected.
To specifically replicate the full length Angiopoietin 2, a primer pair was chosen of
which the upper primer annealed on exon 2, which is not present in the splice
variant. For replication of the Ang-2443, a primer pair was selected, of which the
upper primer annealed to the transition of exon 1 to exon 3, a transition which is not
present in the full length Angiopoietin 2. Primer locations and expected PCR
products are shown in a schematic diagram (figure 2). Primer characteristics are
listed in table 5.
Fig. 2: the primer locations for both primer pairs (black arrows). The thick red line indicates the
replication products in the full length and the splice variant, respectively.
26
Q-PCR primers
(Primer location)
Primer sequence
Optimal annealing
temperature
(Range)
Product
length
Ang-2 full length
Upper
Lower
(376-485)
AGA ACC AGA CTG CCG TGA T
TGT TGT CTG ATT TAA TAC TTG TGC
65 °C (61,4 - 65°C)
110 bp
Ang-2443
Upper
Lower
(293-518)
TACGCA GTG GCT AAT TAA GGT ATT
CTG GAG CTG ATC TTT CTC TTC TTT
64,5 °C
226 bp
Table 5: This table shows the characteristics of the q-PCR primer pairs used for quantification of the
full length Ang-2 and Ang-2443, giving the primer sequences, appropriate temperature ranges,
optimal annealing temperatures, positioning on the gene and product lengths.
For validation of these primer pairs, a temperature gradient was run at
temperatures ranging from 55 °C to 65 °C. Results were analyzed to determine the
optimal annealing temperature and the temperature range within which the PCR
runs properly. PCR products were sequenced and blasted against the canine genome
to determine whether the correct products had been formed. Sequence analysis
showed that the correct products had been formed in both primer pairs.
Quantification of Ang-2 on protein level
To confirm whether the up-regulation of Angiopoietin 2 on mRNA level, as seen in
the q-PCR experiments, was also present on protein level, a Western blot
experiment was performed. In this way, the levels of Angiopoietin 2 protein were
quantified in all of the tumors and normal adrenals, in order to make a comparison
between the levels of Ang-2 protein in normal adrenal glands and ATs.
As a first step, protein was isolated from all of the ATs and normal adrenal glands,
following the protocol for isolation of protein from tissues. After isolation, protein
levels were measured by spectrophotometry using the DC protein assay (BioRad®).
A standard dilution series was made using Bovine Serum Albumin (BSA) in
concentrations ranging from zero to 1.50 mg/ml. Absorbance was measured in all of
the samples and in the standards, using triplicates. From the absorbance in the
dilution series, a standard line was calculated, from which the protein
concentrations in the samples could be deduced. Because the protein concentrations
in the samples were much higher than in the dilution series, all samples were
diluted 25 times in PBS before measurement. Protein concentrations in all of the
samples were sufficient for further analysis by Western Blotting.
27
Protein separation was achieved by performing gel electrophoresis. Because the
molecular weights of the full length Ang-2 and Ang-2443 are 68 and 61 kD
(glycosylated) or 57 and 51 kD (deglycosylated) respectively, for good separation of
both isotypes, the gel was chosen with the highest discriminatory rate between 50
and 75 kD. The gel that fitted these demands best was a 7.5% polyacrylamide gel.
Gels were cast according to the protocol provided by BioRad®, after which gel
electrophoresis was performed. To visualize the progression of electrophoresis and
to allow for accurate estimation of protein size, in each gel a ladder was included,
consisting of dual color Precision plus Protein Standard (BioRad®). After gel
electrophoresis, proteins were transferred onto Hybond® ECL nitrocellulose
membrane.
To prevent aspecific binding of the antibodies to the membrane, blocking of the
membrane was performed using 4% ECL in TBST 0,1%. The primary antibody
consisted of goat polyclonal Ang-2 (C-19): sc-7015, purchased from Santa Cruz
biotechnologies, in a working dilution of 1: 200. Blots were incubated overnight
with the primary antibody, at a temperature of 4 °C.
Initially, the secondary antibody consisted of chicken anti-goat HRP conjugated IgG:
sc-2961, purchased from Santa Cruz biotechnologies. Dilutions of 1: 5000 and 1:
40.000 were used. Blots were incubated with the secondary antibody for one hour
at room temperature. However, no valid results were achieved using this secondary
antibody. Regardless of the antibody concentration and the method of blocking, an
extremely high amount of background staining occurred, making the blots
unsuitable for analysis. Because no solution could be found for this problem, an
alternative secondary antibody was used, consisting of donkey anti-goat HRP
conjugated IgG: sc-2020, also purchased from Santa Cruz biotechnologies. This
secondary antibody was used in a dilution of 1 : 20.000; good results were achieved.
Antibody staining and detection were performed using the ECL advanced Western
blotting detection kit (Amersham RPN2135). Protein detection was performed by
chemiluminescence. For detection, blots were incubated for 2 minutes with the
detection reagent. After incubation, detection was performed using the ChemiDoc®
machine. All blots were first developed for 10 seconds, after which a density
measurement was performed on the bands, to calculate the optimal exposure time.
Blots were then exposed for this period of time to achieve optimal results. Protocols
for protein isolation, gel casting, gel electrophoresis, blotting and antibody staining
and detection are added to this report as an appendix.
To check that the lanes in the gel had been evenly loaded with protein, and to
correct for differences in protein amounts, a loading control was performed, using
Tubulin (Tubulin antibody TUB-1A2, ab11325, AbCam®) of mouse origin. The
antibody was used in a 1: 2000 dilution and the expected band size for this protein
was 55 kD. To perform the loading control experiment, blots were stripped to
remove all antibodies, and subsequently blocked and incubated with Tubulin
antibody for one hour at room temperature. A goat anti-mouse secondary antibody
was used.
To confirm the specificity of the Angiopoietin 2 staining, a blocking peptide for Ang2 was used (Ang-2 blocking peptide, sc-7015P, Santa Cruz biotechnologies). By
incubating the primary antibody solution with an excess of blocking peptide, all
28
antibody is adsorbed to blocking peptide, and thus inactivated, leaving no active
antibody in the solution. Therefore, no staining of the blots should occur. To achieve
this, the primary antibody was dissolved in TBST 0,1% with 4% BSA at a dilution of
1: 200. Blocking peptide was then added in a concentration of 5 times the primary
antibody concentration, and the resulting solution was left to incubate for 2 hours at
room temperature. Blots were stripped to remove all antibodies, and subsequently
blocked and incubated overnight with the pretreated Ang-2 antibody. Protocols for
antibody staining and detection were the same as for the previous Western blot
experiments.
29
Data analysis
Q-PCR data were prepared for analysis using IQ5® software. This software analyses
the raw data produced by the q-PCR, and calculates the Ct value for each sample. The
Ct value is the number of cycles needed for the amount of fluorescence emitted by a
sample to cross a set threshold. This threshold was set to 20 for all of the plates, to
ensure that Ct values were comparable between different plates. A low Ct value
stands for early crossing of the threshold, and thus high expression of the gene in
question.
From the Ct values of the standard dilution series, a standard line is calculated. In
this standard line, the Ct values and the log starting quantity are set out against each
other, resulting in a linear curve; the standard curve. After an ideal q-PCR, the slope
of this curve is -3,321, which gives an efficiency of 100%. To see whether a PCR
reaction had run properly, for each reaction the standard curve was evaluated.
Efficiencies between 95% and 105% were considered acceptable for the reference
genes, whereas for the target genes, efficiencies between 90% and 110% were
considered acceptable. If necessary, deviating Ct values were left out in the
calculation of the standard line to achieve these efficiencies, on the condition that
this change did not cause samples to fall outside the reach of the standard line.
Another focus in preparing the q-PCR data for analysis was the melting curve. This
curve shows the melting temperature of the resulting PCR product, which is
product-specific. If one specific product is formed, the melting curve should show
one single peak, at the temperature expected for that product.
For the initial primer set of Angiopoietin 2, the melting curve consistently showed
an abnormality. For the majority of the samples the melting curve had its peek at the
expected temperature; however some of the samples showed a peek with a different
melting temperature. Some samples also showed a double peek, with both the
normal and the abnormal temperatures. To determine the cause of this abnormality,
gel electrophoresis was run on 6 of the PCR products; two with normal peeks, two
with abnormal peeks and two with double peeks. A double band on the gel was seen
in only one of the samples, in which the abnormal product was shorter than the
normal product. To indentify this abnormal product, sequencing was performed,
and the resulting sequence was blasted against the canine genome. Blast results
showed that the second product was not Angiopoietin related; the sequence
matched part of the sequence of the canine mitochondrion.
These results show that the initial primer pair for Ang-2 has an alternate binding
site, and is therefore not suitable for use in expression analysis of this gene. New qPCR primers were developed, and after validation of these primers, q-PCR for
Angiopoietin 2 was repeated. This time a single peak at the expected temperature
was seen in the melt curve of all of the samples, and data were valid and suitable for
further analysis. The other five target genes also showed qualitatively good data,
which were suitable for expression analysis.
30
To confirm that the reference genes were stably expressed and suitable as reference
genes, the relative expression data of these genes were analyzed using GeNorm®.
This program calculates the stability of gene expression and gives an index which
indicates whether or not a gene is stable enough to serve as a reference gene. An
index below or equal to 1,5 is accepted for a reference gene, whereas a higher index
indicates that the gene is not stable enough to serve as a reference gene. All of the
reference genes used in this study had an index below 1,5 and were thus considered
suitable for use as reference genes.
After preparation, CT values were exported to excel and analyzed using REST-excel,
a program designed for comparing and analyzing q-PCR expression data. This
program operates by comparing two groups, for instance normal controls versus
tumors. The CT values of both target- and reference genes for both groups are
entered into the program. From these values, the program calculates the normalized
expression and then compares this normalized expression between the two groups.
The results of this comparison are given in a fold change; a number that says how
many times the gene is up- or down-regulated in the second group, as compared to
the first. Apart from the fold change, the p-value is calculated, which represents the
chance that the calculated difference between the two groups is based on
coincidence. A p-value below 0.05 was considered significant.
The following groups were compared to each other:
- Normal adrenals versus all tumors (N vs. T)
- Normal adrenals versus adenomas (N vs. A)
- Normal adrenals versus carcinomas (N vs. C)
- Adenomas versus carcinomas (A vs. C)
The version of REST-excel used in this study does not support the use of multiple
reference genes. Therefore, all of the comparisons were calculated separately for
each of the reference genes. Fold changes given in the “Results” section are mean
fold changes, calculated from the REST-excel results for each of the reference genes
in one comparison.
31
Results
Q-PCR for determining expression levels
After q-PCR had been performed on all of the target genes and reference genes, data
analysis was performed using REST-excel. The first step was to check whether all
data were valid and suitable for further analysis. In REST-excel calculations, the
expression levels of one of the reference genes - RPL8 - showed significant downregulation in the tumor-group as compared to the normal controls. The expression
levels of RPL8 showed significant changes in all comparisons, except adenomas
versus carcinomas, with fold changes around 1,5 (Fig. 3).
Fig. 3: In this figure, the changes in expression levels for RPL8 are depicted per category; significant
results are marked with an asterisk. The categories stand for the different comparisons: normal
versus all tumors (N vs. T), normal versus adenomas (N vs. A), adenomas versus carcinomas (A vs. C)
and normal versus carcinomas (N vs. C). On the Y-axis, the fold change is set out; a fold change of 1 or
minus 1 indicates no change in expression levels.
For this reason, RPL8 was excluded from this study as a reference gene, even though
its expression was stable enough in GeNorm. The other three reference genes did
not show any significant changes in expression levels between the tumor- and the
control group, and were therefore suitable for use as reference genes.
As expected, expression of all genes was demonstrated in all of the samples, of both
normal adrenals and adrenal tumors. In the following section the results of
expression analysis will be discussed for each of the genes separately.
Expression analysis of VEGF showed non-significant down-regulation in the whole
tumor group, as well as in the carcinomas, whereas non-significant up-regulation
was seen in the adenomas. When comparing adenomas to carcinomas, VEGF was
32
shown to be significantly down-regulated in the carcinomas, with a fold change of
approximately 8.
VEGFR 1 showed a down-regulation in all of the comparisons, which only reached
significance in the comparison of normal adrenals versus carcinomas. In this
comparison, VEGFR 1 was shown to be down-regulated with a fold change around
2,5. Expression analysis for the last member of the VEGF family, VEGFR 2, did not
show any significant changes in expression levels in any of the comparisons. The
expression changes in the VEGF family are summarized in figure 4.
Fig. 4: This figure summarizes the expression changes in the VEGF family; significant results are
marked with an asterisk. The results are shown per gene, and per category; categories represent the
same comparisons as described for RPL8.
For the Angiopoietin family, expression analysis showed no significant changes in
expression levels of Ang-1 and receptor Tie-2.
In contrast, a clear up-regulation in the tumor-group was seen for Angiopoietin 2,
which was significant in most of the REST-excel calculations for the following
comparisons: normal adrenals versus all tumors (N vs. T), normal adrenals versus
adenomas (N vs. A) and normal adrenals versus carcinomas (N vs. C). Only in the
calculation using RPS5 as a reference gene, significance was not reached, but pvalues were near-significant. Fold changes were comparable for all three reference
genes and for all three comparisons, and ranged between 2 and 2,5. No significant
difference in expression levels was seen between adenomas and carcinomas. The
expression changes in the Angiopoietin family are summarized in figure 5.
33
Fig. 5: This figure summarizes the expression changes in the VEGF family; significant results are
marked with an asterisk. The results are shown per gene, and per category; categories represent the
same comparisons as described for RPL8.
34
Regular PCR for detection of Ang-2443
After development of the primers, the first step was to determine whether or not the
splice variant was present in canine adrenal tissue. To achieve this, PCR was
performed on a sample of normal adrenal tissue, and gel electrophoresis was
performed on the resulting PCR products. The PCR of this sample resulted in the
presence of two bands on the gel, with product lengths of around 550 and 400
basepairs respectively (Fig. 6).
Fig. 6: This photograph shows the results of electrophoresis; the ladder is located on the left side,
and the three columns on the right represent the sample of normal adrenal tissue. On the gel, two
bands are seen, with product lengths around 550 and 400 basepairs respectively.
To confirm that the bands on the gel represented the full length Angiopoietin 2 and
Ang-2443, all bands were carefully cut from the gel. After DNA extraction from the
gel, sequencing was performed, and the resulting sequences were blasted against
the canine genome. The 550 bp product corresponded with the full-length sequence
of Angiopoietin 2 (Fig. 7). The 400 bp product corresponded with two sequences in
the Angiopoietin 2 gene, the first stretch terminating at basepair 311, the second
starting at basepair 468 (Fig. 7). These positions correspond exactly with the
expected sequence of the splice variant, where exon 2 (bp 312-467) should be
missing. These results prove that the splice variant that had previously been
described in man - Ang-2443 - is also present in canine adrenal tissue, and that the
primers designed for detecting this splice variant function properly.
Fig 7: This schematic diagram shows the BLAST results of the 550- and the 400-basepair products,
aligned to the Angiopoietin 2 gene.
35
The next step was to determine whether Ang-2443 was present in all of the samples
used in this study. To achieve this, the same PCR was performed on all of the
samples of adrenal tumors and normal adrenals. PCR products were separated by
gel electrophoresis, and presence of the 550 and 400-basepair bands was evaluated.
Both bands were present in all of the samples, indicating the presence of both the
full length and the alternatively spliced Angiopoietin 2 in all of the tumor and
normal samples (Fig. 8). Band intensities varied considerably among the different
samples. Remarkably, the most intensely stained bands corresponded mostly with
malignant ATs. The intensity of the upper and lower bands, when compared to each
other, also showed variation among the different samples, although the upper band
was always the more prominent of the two.
Fig. 8: This photograph shows the results of electrophoresis ater performing regular PCR for
detection of Ang-2443 on all of the samples. On the photograph, the ladder is located on the upper left
side and on the lower right en left sides, samples 1 through 20 on the upper lane and samples 21
through 40 on the lower lane.
36
Quantification of the full length Angiopoietin 2 and Ang-2443
After validation of both primer sets, and running of the q-PCR, data were analyzed in
the same way as for the previous q-PCR experiments. Preparation of the data for
REST-excel analysis included analysis of the standard curve and melting curve and a
confirmation of the stability of the reference genes, using GeNorm® and RESTexcel®. For all of the reference genes and for the full length Angiopoietin 2, the
standard curve efficiencies were within the accepted limits (95-105 % for the
reference genes and 90-110 % for the targets). However, for Ang-2443 the standard
curve did not reach an efficiency below 125 %. Because REST-excel corrects for the
efficiency, and better results could not be achieved with these primers, the results
were accepted for further calculation in spite of the high efficiency. GeNorm and
REST-excel calculations showed that all three reference genes were stable
throughout the groups and thus suitable for use as reference genes.
After preparation, CT-values for the reference genes and target genes were exported
to excel, and expression analysis was performed using REST-excel. For comparison,
the same groups were used as for the previous q-PCR experiments.
REST-excel analysis showed a significant up-regulation of both the full length
Angiopoietin 2 and Ang-2443 in the comparisons of normal adrenals versus all
tumors (N vs. T), normal adrenals versus adenomas (N vs. A) and normal adrenals
versus carcinomas (N vs. C). Fold changes were consistently around 2 times higher
in the splice variant, indicating that in ATs, Ang-2443 is more strongly up-regulated
than its full length counterpart. Expression levels for both genes tended to be higher
in carcinomas than in adenomas. However, this difference in expression levels was
only significant for Ang-2443. The expression changes for Ang-2 and Ang-2443 are
summarized in figure 9.
Fig. 9: This figure summarizes the expression changes for the full length Angiopoietin 2 and Ang-2443.
Significant differences are marked with an asterisk. The results are shown per gene, and per
category; categories represent the same comparisons as described for RPL8.
37
Quantification of Angiopoietin 2 on protein level
After antibody staining and detection, a prominent band at 68 kD was detected in all
of the samples and a faint 61 kD band in most of the samples. Band positions
correspond with the expected protein sizes for the full length Ang-2 and Ang-2443
respectively. Band intensities vary considerably between samples for both isotypes
(Fig. 10).
Fig. 10: This photograph shows the detection result of one of the blots, with the ladder located on the
right side, and 4 samples located in the columns to the left.
After use of the blocking peptide, both the 68 kD band and the 61 kD band were no
longer visible. Absence of these two bands when blocking peptide is used, shows
that the bands indeed represent Angiopoietin 2; the upper band corresponding with
the full length variant, and the lower band corresponding with Ang-2443.
In part of the samples, a number of aspecific bands were detected in addition to the
specific Ang-2 bands. This might be explained by the fact that a polyclonal primary
antibody was used in this experiment, which often results in more aspecific binding.
After treatment with blocking peptide, these additional bands were no longer
visible, indicating that these bands most likely consist of degrading products of Ang2. The absence of these bands after use of blocking peptide further confirms the
specificity of the antibody.
The loading control experiment using Tubulin antibodies showed presence of the
expected band at 55 kD for all of the samples. Unfortunately, band intensities
differed considerably between different samples, indicating uneven amounts of
protein loaded. Therefore a correction of Ang-2 band intensities was necessary. To
achieve an accurate estimation of Ang-2 expression in each of the samples, the
relative quantities for both Ang-2 and Tubulin, as given by the Quantity One®
program on the ChemiDoc, were normalized to the exposure period in seconds.
38
After normalization, Ang-2 band intensities were corrected for Tubulin band
intensities in the corresponding samples.
For the comparison of expression levels, two methods were used separately. The
first method was a semi-quantitative method, in which band intensities for Ang-2,
Ang-2443 and Tubulin were scored. Possible scores ranged from – (no band visible),
± (band visible, but with difficulty) to +++ (very thick band with high intensity).
After scoring of the bands, results were compared between the following groups:
normal controls (N), adenomas (A), carcinomas (C) and the whole tumor group (T).
Results of this method of comparison showed that expression levels of Ang-2 were
consistently much higher than those of Ang-2443. Both variants were present in
higher amounts in the tumor groups, and for Ang-2443 a difference between
adenomas and carcinomas seemed to be present.
Because of the inaccuracy of this semi-quantitative assessment, a second method of
comparison was also applied, in which the relative quantities as measured by the
Quantity One® program were used for comparing the expression levels. For this
method, the relative quantities as given by the Quantity One® program and
corrected for Tubulin staining as described above, were grouped and means were
calculated for each group. Groups were the same as for the first method of
comparison. Results are shown in figures 11 and 12.
Fig. 11: This figure shows the expression changes on protein level for the full length Ang-2. The
normalized and corrected band intensities are depicted on the Y axis and represent the expression
level of the protein.
39
Fig. 12: This figure shows the expression changes on protein level for Ang-2443. The normalized and
corrected band intensities are depicted on the Y axis and represent the expression level of the
protein.
The expression levels of the full length Ang-2 protein are considerably higher in the
tumor group, in comparison to the normal controls. No difference is seen between
adenomas and carcinomas (Fig. 11). Expression levels of Ang-2443 protein are
generally much lower than those of the full length variant. However, like the full
length Ang-2, Ang-2443 shows markedly higher expression levels in the tumor
groups as compared to normal controls. Furthermore, for Ang-2443, a clear
difference in expression levels is present between adenomas and carcinomas, with
higher expression levels in the malignant tumors (Fig. 12). These results are in
correspondence with the results of the semi-quantitative scoring method.
40
Conclusions
From the results of this study, a number of conclusions can be drawn, with regard to
the expression of the target genes in ATs. Of the six target genes, Angiopoietin 2
showed the most promising results. Expression analysis of the q-PCR results
showed significant up-regulation of Ang-2 mRNA in the ATs, when compared to
normal adrenal tissue. Additionally, by means of regular PCR and sequencing, the
presence of Ang-2443, a splice variant of Ang-2, which misses exon 2, has been shown
in both ATs and normal adrenal glands. This splice variant had previously been
described in man, and has been linked to tumorigenesis in some human studies. To
my best knowledge, this is the first study in which this splice variant has been
described in the canine species.
Expression analyses of the full length Ang-2 and Ang-2443 have been performed, to
see whether the splice variant is differentially expressed between normal adrenal
tissue and ATs. Results of this analysis show that both the full length Ang-2 and Ang2443 are significantly up-regulated on mRNA level in the ATs. Fold changes were
consistently around two times higher in the splice variant than in the full length,
indicating that the splice variant is more strongly up-regulated in these tumors than
the full length Ang-2. Another interesting outcome was that unlike the full-length
Ang-2, Ang-2443 showed a clear up-regulation in carcinomas as compared to
adenomas.
To confirm the q-PCR findings, a Western blot experiment was performed, to
quantify and compare the expression levels of both the full length Ang-2 and its
splice variant, on protein level. Results of this experiment show a clear upregulation on protein level of both the full length Ang-2 and Ang-2443 in the tumor
groups, when compared to normal controls. For the full length Ang-2, no difference
in expression levels between adenomas and carcinomas was seen, whereas for Ang2443 markedly higher expression levels were seen in the carcinomas. The Western
blot results thus confirm the q-PCR findings regarding the expression of Ang-2 and
Ang-2443 in ATs.
Both q-PCR and Western blot results thus strongly indicate a role of Ang-2 in the
pathogenesis of ATs in dogs. Because both the full length Ang-2 and Ang-2443 are
over-expressed in ATs, it seems likely that both isotypes are involved in tumor
pathogenesis. The stronger up-regulation of Ang-2443 and its differential expression
between adenomas and carcinomas might indicate a specific role of this splice
variant in the pathogenesis of ATs.
For the other genes, the results of expression analysis were less prominent. No
significant changes in mRNA expression levels were detected for Ang-1, Tie-2 and
VEGFR 2. These results indicate that Ang-1, Tie-2 and VEGFR 2 are most probably
not involved in ATigenesis in dogs. Expression analysis of VEGF and VEGFR 1
showed no significant changes in most of the comparisons. However, VEGF was
significantly down-regulated in the comparison of carcinomas versus adenomas (C
vs. A) and VEGFR 1 was significantly down-regulated in the comparison of normal
adrenals versus adenomas (N vs. A). The roles of VEGFR 1 and VEGF are in canine
41
AT pathogenesis are therefore still unclear, as down-regulation was in both cases
only present in one of the comparisons and no changes were detected in the other
comparisons. Furthermore, in the case of VEGF, down-regulation is not the expected
result in case of a contribution to tumor pathogenesis of this gene. Therefore, more
research is needed in order to draw valid conclusions about a possible role of these
genes in canine AT pathogenesis.
42
Discussion
Before discussing the actual results of expression analysis, some general remarks
are in place about the materials en methods used in this study.
The first point of discussion concerns the method of tumor classification. As
previously described, for comparison a distinction was made between normal
adrenal glands, the whole tumor group, adenomas and carcinomas. The expression
data within these groups were compared to each other, to detect differences in
expression levels between the groups. The distinction between adenomas and
carcinomas was based upon histological evaluation of the tumors and upon clinical
data regarding metastasis. However, the classification of canine ATs raises some
difficulties. A differentiation between benign and malignant tumors is often difficult,
when no obvious metastases or invasive growth can be detected. As a result,
different pathologists may come to different classifications on histological
evaluation.
To overcome this problem in the present study all the tumors were evaluated by
one single pathologist. The tumor was regarded malignant when at least one or
more of the following criteria were met: the presence of metastasis, vascular
ingrowth and capsular invasion by the tumor cells. Tumors that lacked these three
criteria were characterized as adenomas. This classification will most likely be
reliable for most of the tumors, but the reliability would be greatly enhanced by
using and combining more criteria. Therefore, caution is needed in the
interpretation of expression analysis based on this tumor classification.
In a recent publication on the indicators of malignancy in canine ATs, a large
number of different criteria were assessed for their potential in AT classification66.
This assessment resulted in a list of criteria, with a significant association with
either adenomas or carcinomas. Significant indicators of malignancy in ATs were: a
diameter over 2 cm, peripheral fibrosis, capsular invasion, trabecular growth
pattern, hemorrhage, necrosis, and single-cell necrosis. Significant indicators of
benign ATs were: hematopoiesis, fibrin thrombi, and cytoplasmic vacuolation. Apart
from these morphologic characteristics, the potential of the immunohistochemical
proliferation marker KI-67 in distinguishing benign and malignant ATs was also
evaluated. This evaluation showed, that a high KI-67 proliferation index was
significantly correlated to malignancy, and a threshold value of 2,4 would correctly
classify 96% of all ATs. Thus, for AT classification, a combined evaluation of these
morphological criteria and a KI-67 staining would produce the most reliable results.
Re-assessing the ATs in that way would thus be a valuable recommendation for
improving the reliability of the results of expression analysis.
A second point of discussion is the amount of angiogenesis itself. As explained in the
introduction, angiogenesis plays an important role in the pathogenesis of many
tumors. For this reason, genes that are involved in the regulation of angiogenesis
were chosen as target genes for this study. In the hypothesis that genes involved in
angiogenesis would be up-regulated, the presumption is made that angiogenesis is
43
also more active in these ATs: if no increase in angiogenesis would be present, no
up-regulation of any of these genes would be expected.
The results of this gene expression study, especially the up-regulation of Ang-2,
indicate that angiogenesis may indeed be increased. However, this cannot be
concluded from the results of expression analysis alone. To confirm the involvement
of increased angiogenesis in the pathogenesis of these tumors, an evaluation of the
vascularity of these tumors should be performed. This would also strengthen the
conclusions of this study. A difference in expression levels of some genes involved
in angiogenesis is obviously present and the involvement of these genes in AT
pathogenesis is therefore likely; however, without an evaluation of the vascularity of
the tumors, no definitive conclusions can be drawn concerning the connection
between the expression differences and actual angiogenesis. If an evaluation of the
vascularity of the tumors were available, the correlation between increased
vascularity and increased gene expression could be calculated, and a conclusion
could be drawn concerning the relation between those two variables. Such an
evaluation would thus be a valuable recommendation for future research.
Another point of discussion is the number of tumor- and normal samples used in
this study. As described in material and methods, 31 ATs were evaluated, along with
9 normal adrenals. In an ideal situation, these groups should be of approximately
the same size, so that a better comparison can be made. In this study, the negative
effect of unequal group sizes could be seen in some of the comparisons. In these
comparisons, a trend is visible in the expression levels, which does not reach
significance, but might do so if the number of normal controls were doubled.
Therefore, repeating the q-PCR analyses using more normal controls, so that equal
group sizes are achieved, would considerably strengthen the results of this study.
To gain more insight in the influence of ACTH in the regulation of target gene
expression, an ACTH-stimulation experiment was performed. However, in this
experiment no differences between the stimulated group and the control samples
could be detected, in either expression levels of the target genes, or cortisol
concentrations. The absence of a rise in cortisol levels in the ACTH treated group
shows that no ACTH stimulation has occurred in the tissue fragments. Therefore,
from this experiment no conclusions could be drawn about the regulating effect of
ACTH stimulation on the target genes, and the results of this experiment were thus
left out of this report.
However, as previously mentioned, ACTH has been reported to influence most of the
target genes investigated in this study and ACTH levels are known to be extremely
low in dogs with functional ATs. These low ACTH levels might well influence the
target gene expression in these dogs, and thus the results of this study. Therefore, it
may be a valuable addition to repeat this stimulation experiment, using multiple
samples of both normal adrenal tissue and ATs and looking carefully at the
conditions for this experiment. In this way, the influence of ACTH on the expression
levels of all of the genes of interest in the canine adrenal gland can be made clear.
44
For the Western blot experiment, a note of discussion concerns the amounts of
protein loaded to the gel for electrophoresis. As described in the “Results” section,
the loading control experiment did not result in equal amounts of Tubulin staining
for each of the samples. This indicates that uneven amounts of protein have been
loaded to the gel. The explanation for this uneven loading may lie in a number of
different problems, for example inaccuracies in the protein measurement, pipetting
errors or inadequate mixing of the dilutions for protein measurement. Another
possible explanation lies in the homogeneity of the protein samples. In some of the
samples, the protein lysate does not appear to be entirely homogeneous and
although all samples were mixed before pipetting, this may still have led to
differences in the amount of protein loaded for each sample.
To minimize the negative effects of the unequal protein loading, two methods of
comparison were applied, as described in the “Results” section; a semi-quantitative
method involving scoring of the band intensities, and a method using the relative
quantities as calculated by the ChemiDoc® software. For the last method, all
Western blot results were normalized for the exposure time and subsequently
corrected for Tubulin quantities. In this way the relative quantities could still be
compared to each other in spite of the differences in the amounts of protein loaded.
Both methods gave roughly the same results, which increases the reliability of these
results. However, better and more reliable results would be achieved by repeating
the experiment, and making sure that the amount of protein loaded is the same for
each sample. If further research were to be done on this subject, repeating this
experiment that way might thus be a valuable recommendation.
The results of the q-PCR, regular PCR and Western blot experiments performed in
this study revealed some interesting, and sometimes unexpected, outcomes. In the
next paragraphs a discussion of these results will follow for each of the target genes
separately, taking into account the expectations, based on literature research, and
examining potential explanations for unexpected results.
VEGF
Based on the literature, an up-regulation of VEGF in the tumor group was expected,
as VEGF is known to be up-regulated in many different types of tumors, both in man
and in dogs. Additionally, in many studies, a correlation was shown between VEGFlevels and malignancy, in which malignant tumors tended to have higher levels of
VEGF expression16,21,31,38,13,38,40,42. However, the results of expression analysis do not
show an up-regulation of VEGF in the ATs investigated in this study. Instead, a nonsignificant down-regulation is seen in most comparisons, and even a significant 8fold down-regulation when comparing carcinomas to adenomas. Another
interesting result is that although VEGF is either not significantly changed or even
down-regulated, Angiopoietin 2 is significantly up-regulated. In the literature, these
factors often show a significant correlation; both are up-regulated together17,19,22.
Also, VEGF is an important stimulatory factor of Ang-2 expression; high levels of
VEGF induce an increase in Ang-2 expression31. Therefore the strong up-regulation
of Ang-2, while VEGF remains unchanged or is even down-regulated, is a remarkable
outcome.
45
One possible explanation for the absence of the expected up-regulation of VEGF is
that there is a difference in the behavior of VEGF between different tumor types
and/or different species. Although raised VEGF levels have been shown in various
tumors, both in man and in dogs, no studies have been performed concerning VEGF
levels in ATs in either species. Possibly, VEGF behaves differently in these tumors; a
difference between canine and human patients is also a possibility.
Another possible explanation may be found in the type of tumor used in this study,
and the regulation of VEGF expression. In this study, only functional ATs are
investigated; in other words, tumors from dogs suffering from Cushing’s syndrome .
In these dogs, the negative feedback mechanism results in a strong decrease in
ACTH levels. This decrease might influence VEGF levels, as ACTH is one of the
factors known to up-regulate VEGF expression, and a study in mice showed that its
depletion in iatrogenic hypercortisolism led to a decrease in VEGF levels, which
could be reversed by ACTH supplementation56.
However, these theories still do not provide an explanation for the discrepancy in
the behavior of VEGF and Angiopoietin 2. In order to find possible explanations, a
literature search was performed, looking for factors that positively affect Ang-2
expression, but have no influence – or a negative influence – on VEGF expression. In
the figure 13, a schematic representation is given of the regulatory mechanisms of
the expression of both genes.
Fig. 13: In this diagram, a schematic representation is given of the regulatory mechanisms involved
in the expression of VEGF and Ang-2. Factors that elevate Ang-2 expression, but have no influence (or
a negative influence) on VEGF expression, are depicted in green.
46
An interesting candidate to explain the discrepancy in behavior of both factors is
glucose. High glucose levels induce Ang-2 expression, while VEGF expression is
down-regulated in vivo in response to hyperglycemia64,65 (Fig. 13). As explained in
the introduction, the high levels of glucocorticoids produced in dogs with Cushing’s
syndrome , cause an increase in gluconeogenesis, and consequently an increase in
blood glucose levels. That makes glucose an interesting candidate to explain the
difference in behavior between VEGF and Ang-2. However, it needs to be mentioned
than none of the dogs had suffered from diabetes mellitus.
Apart from glucose, some other factors can be identified from the regulation
diagram that up-regulate Ang-2, but have no effect on VEGF expression. These
factors include for instance PI 3, HER 2 and basic fibroblast growth factor and are
depicted in green in the diagram. To identify which factor actually causes the
discrepancy, more research is needed. Recommendations for further research might
include q-PCR for the factors that positively influence Ang-2, but not VEGF, and
analysis of the plasma glucose levels in the patients from which the tumors were
excised, to see if a correlation between glucose levels at the time of surgery and the
expression levels of both genes can be found.
The strong down-regulation of VEGF in adrenocortical carcinomas is more difficult
to explain, as in the literature in many cases a positive correlation was shown
between VEGF levels and malignancy, and to my best knowledge, no studies have
yet reported a down-regulation of VEGF in malignant tumors. Further research is
therefore needed to ascertain that VEGF is indeed down-regulated in canine
adrenocortical carcinomas, and if confirmed, to determine possible mechanisms and
causes of this down-regulation.
VEGFR 1
Expression analysis shows a down-regulation of VEGFR 1 mRNA, which reaches
significance only in the comparison between normal adrenals and adenomas. These
results contradict our hypothesis that VEGFR 1 would be up-regulated in ATs.
However, as was discussed in the introduction to this gene, the exact role of this
receptor is still subject to discussion, and although VEGFR 1 has been shown to be
up-regulated in some studies32,33,37,49, this up-regulation was not consistently
present in different kinds of tumors. A tumor-type dependent behavior of the
expression levels of this gene is therefore likely. On the expression levels in ATs, to
my knowledge no information was available in either man or dog.
Furthermore, the predominant theory on the function of VEGFR 1 is that it acts as a
decoy receptor, preventing the binding of VEGF to VEGFR 2. In this theory, VEGFR 1
would act as a negative regulatory receptor for angiogenesis. Down-regulation of
this receptor would therefore be in line with our hypothesis that angiogenesis is
increased in ATs, and genes stimulating angiogenesis should be up-regulated,
whereas genes inhibiting angiogenesis should be down-regulated.
VEGFR 2
Based on the role of VEGFR 2 as the primary mediator of VEGF’s stimulatory action
on angiogenesis, and on its up-regulation in some tumors in the literature, our
hypothesis was that this gene would be up-regulated in the tumors. However, the
47
results of this study do not correspond with the hypothesis, as VEGFR 2 does not
show any difference in expression levels in any of the comparisons made in REST
excel.
As for VEGF, there are a few possible explanations for these unexpected results.
First, an increase in VEGFR 2 expression levels in tumors has only been shown in
some studies, whereas in other studies no changes in VEGFR 2 levels were seen.
Therefore, the behavior of VEGFR 2 may differ, depending on the tumor type and
species, like the behavior of VEGFR 1. Unfortunately, no information on VEGFR 2
expression levels in ATs was available.
Another possible explanation lies in the regulatory mechanism of VEGFR 2. As for
VEGF, expression of this receptor was shown to decrease in response to low levels
of ACTH, as was shown in mice with iatrogenic hypercortisolism56. As ACTH
decreases to undetectable levels in dogs with Cushing’s syndrome as a result of the
negative feedback mechanism, a lack of ACTH stimulation might explain the low
levels of VEGFR 2.
Finally, as VEGFR 2 is the primary mediator of VEGF action, it may not be surprising
that both behave in the same way. The combination of normal to low VEGF levels
and unchanged VEGFR 2 levels may simply indicate that the VEGF pathway is not
active in these tumors. Further research is needed to determine whether the VEGF
pathway is involved in canine AT pathogenesis, and if so, in what way it is involved.
Angiopoietin 1
As described in the introduction to this gene, the formulation of a hypothesis
regarding the behavior of Ang-1 in canine ATs was difficult, as contradicting
information exists on its expression in tumors14,29,30. Therefore our hypothesis was
based on the knowledge of the function of this gene: Ang-1 acts as a stabilizing
factor on the vascular endothelium, whereas in most tumors with active
angiogenesis a destabilized vascular endothelium is observed. Also, in many tumors
this destabilization is observed in combination with increased vascular
permeability, whereas Ang-1 functions in decreasing vascular permeability and
protecting against vascular leakage6,7,9,10-12. For these reasons, we expected to find
down-regulation of Angiopoietin 1 in canine ATs.
However, the results of this study contradict our hypothesis, as no significant
differences in expression levels were seen in any of the comparisons for
Angiopoietin 1. A possible explanation for this unexpected result may be that as
Ang-1 and Ang-2 function as competitive antagonists, the amount of angiogenesis
and the stabilization or destabilization of the vascular network is a result of the
balance between these two genes. A raise in Ang-2 levels might thus be sufficient in
changing the balance towards destabilization and angiogenesis, without a decrease
in Ang-1 being present. To test this hypothesis and to determine whether or not
Ang-1 is involved in canine AT angiogenesis, further research is necessary.
Angiopoietin 2
As described in the introduction to this gene, Angiopoietin 2 is a regulatory protein
in angiogenesis, which causes a destabilization of the vascular endothelium. In the
48
presence of VEGF, this destabilization precedes proliferation and migration of
endothelial cells and vessel sprouting, resulting in increased angiogenesis15.
Because of these features, a role of Ang-2 in tumor pathogenesis in general and in
tumor angiogenesis in particular was deemed likely. Indeed, numerous studies in
both man and dog, have shown elevation of Ang-2 expression in different kinds of
tumors, including human ATs3-5,16-20,22,30. In many cases this elevation was shown to
correlate to increased vascularity, and often a correlation with tumor grade was also
shown16,17,21,22. Based on these findings, the hypothesis was formulated that Ang-2
would also be involved in the pathogenesis of the canine ATs investigated in this
study, and levels of Ang-2 expression in these tumors would be higher than those in
normal adrenal glands.
In accordance with this hypothesis, expression analysis performed on the ATs in this
study, showed a clear up-regulation of Ang-2 on mRNA level, which was significant
in almost all comparisons. To determine whether this up-regulation was also
present on protein level, a Western blot study was performed. Results of this study
showed a clear up-regulation of Ang-2 protein in the ATs, thereby confirming the qPCR results.
As already discussed, the presence of VEGF is needed for Ang-2 to play a role in
stimulation of tumor angiogenesis. Although VEGF was not over-expressed in the
ATs used in this study, it was present in all of the samples, thus enabling the
angiogenesis-promoting role of Ang-2. These results therefore support the
hypothesis that increased expression of Ang-2 contributes to tumor angiogenesis in
ATs in dogs, and in that way plays a role in the pathogenesis of these tumors. To
further strengthen this theory, it would be worthwhile to perform histological
examination of all of the tumors, in which the vascularity is assessed. A correlation
between elevated levels of Ang-2 and increased vascularity would provide further
evidence that Ang-2 is indeed involved in AT pathogenesis by influencing tumor
angiogenesis. Another useful recommendation for further research on this subject
would be to perform immunohistochemistry, in order to indentify the exact
localization of Ang-2 production within ATs.
Another interesting outcome of this study was the existence of Ang-2443, a splice
variant of Ang-2, which differs from the full length variant by missing exon 2. As
discussed in the introduction, this splice variant had previously been identified in
man and some studies have indicated a role in human tumor pathogenesis for this
variant61,62. Because to our best knowledge, no information was available on the
presence, or the role in tumor pathogenesis, of Ang-2443 in dogs, this study aimed to
investigate the presence of this splice variant and if present, to quantify its
expression.
As discussed in the “Results” and “Conclusions” sections, in this study the presence
of Ang-2443 in canine ATs and normal adrenal glands was confirmed by means of
regular PCR, and its expression quantified by means of q-PCR and Western blot.
Expression analysis showed an increased expression of both the full length Ang-2
and Ang-2443 in the investigated tumors on both mRNA level and protein level.
These results are in line with our hypothesis, and with the up-regulation of Ang-2 as
seen in the previous q-PCR experiments. An interesting outcome was that the fold
changes were consistently around twice as high in Ang-2443 than in the full length
49
variant; Ang-2443 was thus up-regulated to a higher extend than the full length Ang2. Another interesting outcome was that unlike the full length Ang-2, Ang-2443
showed a clear difference in expression levels between adenomas and carcinomas,
both on mRNA and on protein level. These results might indicate a specific role of
Ang-2443 in the pathogenesis of these tumors, and are consistent with the specific
up-regulation of this splice variant in some tumors in man.
However, which specific role Ang-2443 might play in tumor angiogenesis is still
unclear, as studies in humans showed that Ang-2 and Ang-2443 most likely function
in the same way, as a competitive antagonist of Ang-1. Further research is needed to
confirm a specific role in tumor angiogenesis of Ang-2443, and to determine the
nature of this role.
Tie-2
Based on the functions of Tie-2 and its raised expression in a variety of human
tumor types, an up-regulation of this gene in the ATs was expected. However, in this
study, no change in Tie-2 expression was detected in any of the comparisons.
A possible explanation for this discrepancy lies in the regulation of Tie-2 expression.
As for VEGF, VEGFR 2 and Ang-1, regulation of Tie-2 expression is ACTH dependent.
A study showed that Tie-2 expression decreased in mice with iatrogenic
hypercortisolism, which could be reversed by supplementing ACTH55. Because of
the negative feedback mechanism, ACTH concentrations in dogs with corticosteroid
producing ATs are extremely low, which may explain why no rise in Tie-2 levels was
seen in these tumors. Another possibility is that the behavior of Tie-2 depends on
the tumor type or species, and that over-expression of this gene is simply not
involved in canine AT pathogenesis. To determine which of these theories is the
most likely, more research is needed.
50
Acknowledgements
Coming to the end of this paper, I would like to thank all the persons who have
contributed to this study. Many thanks I owe to my supervisors Jan Mol, Hans
Kooistra and Sara Galac, for their continuing guidance, support, input and
suggestions, and for making it possible for me to stay just a few months longer and
thus enabling me to really finish this project, instead of just handing it over to the
next student. I would also like to thank Elpetra Sprang-Timmermans, Monique van
Wolferen and Adri Slob for their great help and guidance in the laboratory work and
their continuing input, without which I could never have done any of this. The other
research analysts also deserve a thank you, for their help in the lab and for always
answering my many questions. The same goes for the PhD students, especially Ana
and Gaya for their help and suggestions, both during the weekly meetings and in the
lab. And last but not least all of the other students and employees at the department,
for their companionship and support, and for making the past nine months not only
an extremely interesting research period, but also al lot of fun.
51
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